Most NF1 patients (>90%) develop tumors within the peripheral ganglia, peripheral, and/or cranial nerves called neurofibromas, composed of cell types including neuronal axons, fibroblasts, perineurial cells, Schwann cells, and mast cells (7). Homozygous loss of NF1 is present only within the Schwann cell compartment (8–10) indicating that cells within the Schwann cell lineage are necessary for neurofibroma formation. In humans, plexiform neurofibromas can be congenital, suggesting a possible role for a developing Schwann cell in neurofibroma formation. However, the cell(s) of origin for neurofibroma formation within the Schwann cell lineage remain unclear.

Neural crest cells develop into Schwann cell precursors between E11 and E13 in mouse sciatic nerve, and Schwann cells by E18 (11–12). Progenitors identified after the establishment of the dorsal root ganglia (DRG) have more limited self-renewal and differentiation potential than neural crest stem cells (13–17). Schwann cells differentiate in close association with the axons of peripheral nerves. Those Schwann cells associated with neuronal cell bodies form satellite cells that express either S100β or GFAP. Schwann cells ensheathing multiple small axons in peripheral nerves are GFAP+ nonmyelinating Schwann cells, whereas Schwann cells associating with single large axons form myelin and express S100β and periaxin (18). Neurofibroma initiating cells may be committed glial cells, de-differentiated Schwann cells, and/or post-crest progenitor cells.

Nf1−/− mouse embryos die by E13.5 due to abnormal heart development and Nf1+/− mice do not develop neurofibromas (19–20). Loss of Nf1 in animal models using neural crest drivers Wnt1-Cre (E9.5), Mpz–Cre (E9.5–10.5), and Pax3–Cre (E10.5) did not result in neurofibroma formation (21). These findings suggested that a post-crest target cell(s) drives neurofibroma formation; therefore, several laboratories targeted Nf1 loss to Schwann cell populations after neural crest migration. In a pioneering study a Krox20–Cre driver line, which expresses within boundary cap cells at E10.5 and later in Schwann cells in peripheral nerves, caused genetically engineered mice (GEM) neurofibroma formation (22). A P0A-Cre+ driver line in which loss of Nf1 begins in neural crest at E9.5 with robust expression at E12.5 also enabled neurofibroma formation (23). Loss of Nf1 in embryonic Schwann cells (E12.5) also caused neurofibroma formation (24). Thus cell(s) developing at or after the embryonic Schwann cell stage of development initiate neurofibroma formation.

Peripheral nerve Remak bundles containing small diameter axons ensheathed by a single Schwann cell are disrupted in all neurofibroma models. In contrast, myelinated axons appear relatively spared. This led to the suggestion that nonmyelinating Schwann cell are tumor-initiating cells within neurofibromas (23).

Myelin proteolipid protein (Plp) is a component of the Schwann cell myelin sheath (25). We used a tamoxifen-inducible PlpCre driver line [Plp-Cre-ERT (designated PlpCre throughout)] to test the potential role of Nf1 loss within Plp-expressing cells in neurofibroma formation (26), inducing Nf1 loss after birth or in adult animals. We report that Nf1 inactivation at either age results in neurofibroma formation. While Remak bundle disruption is shown within the neurofibromas, GFAP+ nonmyelinating Schwann cells do not show Nf1 inactivation.

Materials and Methods

Mouse husbandry

Cincinnati Children's Hospital Research Foundation animal care and use committee approved all animal use. Mice were housed in a temperature- and humidity-controlled vivarium on a 12-hour light-dark cycle with free access to food and water.

Perinatal tamoxifen injections

Tamoxifen (100 mg) was dissolved in 1 ml of ethanol and 9 mls of sunflower seed oil. Intraperitoneal (i.p.) tamoxifen injection (1 mg/100 μL) was twice a day for 3 consecutive days to lactating mothers, administering tamoxifen to pups through the mother's milk, beginning when pups were 1 day old. Dose-limiting toxicity was pup trembling and occasional mortality, when tamoxifen was provided twice a day for >3 days. Tamoxifen injection once daily for 3 or 5 days to the mother failed to cause peripheral nerve recombination.

Adult tamoxifen injections

One hundred microliters (1 mg/100 μL) was administered i.p. once-or-twice-a-day for 3 consecutive days. Tamoxifen administration once daily for 3 or 5 consecutive days did not result in significant adult peripheral nerve recombination. We dosed twice a day for 3 consecutive days.

Genotyping and recombination and survival studies

Mice were genotyped by PCR (22, 26, 27). Nf1 recombination was determined 30 days after tamoxifen injections using PCR (22). PlpCre;Nf1fl/fl mice were euthanized when they became paralyzed, failed to groom, had obvious weight loss, or developed tumor masses.

Tissue processing

We administered Brdu i.p. (50 mg/kg body weight) 3 times at 2-hour intervals. Two hours later, we anesthetized mice and perfused with ice cold 4% paraformaldehyde. Tissues were removed and photographed on a Leica MZFL111 microscope, then post-fixed in 4% paraformaldehyde overnight for paraffin sectioning or for an hour with transfer to 20% sucrose for frozen sectioning. For electron microscopy, we perfused mice with 4% paraformaldehyde and 2.5% glutaradehyde, post-fixed in the same fixative overnight, and then transferred tissues to 0.175 mol/L cacodylate buffer, osmicated, dehydrated, and embedded in Embed 812 (Ladd Research Industries). Ultrathin sections were stained in uranyl acetate and lead citrate and viewed on a Hitachi Model H-7600 microscope.

Histology

Paraffin sections were processed for H&E to examine tissue structure or stained with Toludine Blue to identify mast cells. An in vitro Brdu staining kit monitored proliferation (Invitrogen). Biotinylated secondary antibodies were used at 1:200 (Vector), together with an ABC kit to visualize immunoreactivity (Vector Labs).

Statistical analyses

Kaplan–Meier survival curves were created using GraphPad Prism software and Log-rank Mantel–Cox Tests. Counting of Schwann cells and satellite cells was carried out on at least 150–300 EGFP+ cells per area per animal from 3–5 animals per genotype. Two-way t-tests were carried out on complete blood count (CBC) X Blood Smear counts and immuno-reactive cell counts with a significance cutoff of P < 0.05.

Results

Postnatal (P1–3) loss of Nf1 in PlpCre;Nf1fl/fl mice causes early and later mortality

The remaining PlpCre;Nf1fl/fl animals (29/36; 80%) required sacrifice between 15 and 21 months of age. Littermate PlpCre;Nf1wt controls (designated “WT controls” or wild type throughout the manuscript) remained healthy. On gross dissection these PlpCre;Nf1fl/fl animals had enlarged peripheral nerves associated with paraspinal tumors at cervical (Fig. 1B) and thoracic spinal levels. Sixty-seven percent (4 of 6) of mice in which full neuroaxis dissection was carried out also had paraspinal tumors within the lumbar/sacral regions (Supplementary Table S1). Grading and classification of tumors used GEM nerve sheath classification (28). GEM neurofibromas Grade I are defined as tumors with histological features similar to those of human neurofibromas; hypocellular with abundant matrix and collagen fibers, minimal cell atypia and rare mitosis. There are often intermixed nerve axons and mast cells present. All paraspinal tumors in the PlpCre;Nf1fl/fl model were GEM Grade I neurofibromas. On histological analysis, they showed low cellularity, stromal matrix between the cells, S100β+ myelinating Schwann cells, and mast cell infiltration (Fig. 1C).

Endogenous Plp is expressed in spleen and thymus as well as glial cells (29). Therefore, this PlpCre driver could be expressed within hematopoietic cell populations. Postnatal (P1–3) tamoxifen injections within PlpCre;Nf1fl/fl animals caused development of tumors resulting from extramedullary expansion of lymphoid and myeloid cell populations in 36% of the animals examined. Seven developed early (by 5 to 7 months of age) and 7 animals contained hematopoietic-containing tumors at necropsy in the context of complications arisen from neurofibroma formation (15 to 22 months of age). Enlarged organs with solid white lesions were noted within the liver, spleen, lung, kidney, lacrimal gland, and lymph nodes within the abdominal cavity, neck, inguinal, and axillary regions (Fig. 2A). Within the subset of animals that developed these tumors, splenomegaly, a prominent finding in a number of hematopoietic abnormalities including JMML, was common (Fig. 2A, right). These animals often suffered dermatitis of the face and neck. While eczema is common in JMML, these dermal lesions overlaid neurofibromas deep within the tissue.

Gross dissections were carried out on the neuroaxis of 20 PlpCre;Nf1fl/fl animals. All had tumors associated with peripheral nerves and DRGs (Supplementary Table S1), and cranial-facial nerves. Figure 3B displays representative tumors associated with spinal and peripheral nerves of a PlpCre;Nf1fl/fl animal, diagnosed as GEM Grade I neurofibromas after paraffin embedding and staining with H&E (Fig. 3C). Similar to perinatal PlpCre;Nf1fl/fl neurofibromas, these displayed low cellularity with increased stroma between the cells. The neurofibromas contained characteristic S100β+ cells and mast cell infiltration (Fig. 3C).

Recombination was examined by PCR analysis (Fig. 3D). As expected, recombination was noted within the brain and peripheral nervous system (sciatic nerve and dorsal root ganglia). Recombination was also detected in the heart, lung, spleen, thyroid, skin, fat, and bone. Recombination was absent within the liver, kidney, and bone marrow. EGFP+ cells were present within peripheral nerves throughout the body (data not shown). Organ-specific EGFP+ cells were also noted within the heart, lung, thyroid, and spleen (Supplementary Fig. 2), likely accounting for some of the recombination observed.

Comparison of neurofibroma formation after perinatal or adult tamoxifen exposure in PlpCre;Nf1fl/fl mice

Remak bundle disruption within PlpCre;Nf1fl/fl saphenous nerves

Electron microscopy compared saphenous nerves of 12-month-old PlpCre+;Nf1wt control (Fig. 5A), postnatal (P1–3) tamoxifen-injected PlpCre+;Nf1fl/fl (Fig. 5B), and adult tamoxifen-injected PlpCre+;Nf1fl/fl animals (Fig. 5C) to determine whether loss of Nf1 within PlpCre+ cells affects Remak bundles. Similar to other mouse neurofibroma models, myelination of large diameter axons appeared normal after either perinatal or adult loss of Nf1 whereas perinatal or adult tamoxifen exposure caused Remak bundle disruption (Fig. 5B & C). Thus many small caliber axons were found in a one to one relationship with an attendant axon while other Schwann cells were dissociated from axons completely, and pathologically wrapped collagen.

EGFP+ cells after tamoxifen injection in PlpCre+ animals. A, graph (left) indicates percentages of EGFP+/DAPI+ cells in sciatic nerve and DRG/satellite cells and (right) the percent of EGFP+ recombined cells that double labeled with S100β, GFAP, p75, or GS. B, double immunolabeling (40×) shows EGFP+/S100β+ cells within the DRG or sciatic nerve one-day-post Tamoxifen. C, EGFP+/GFAP+ cells are present in the DRG but not sciatic nerve. D, EGFP+/p75+ cells are absent in the DRG and present in sciatic nerve. Immunohistochemistry in neurofibromas after adult tamoxifen exposure within the PlpCre;Nf1fl/fl model shows EGFP double labeling with S100β+ (B) and p75 (D) but not GFAP+ (C).

In wild type mice, the percentage of double-labeled EGFP+;GFAP+ cells 4 days after tamoxifen within the DRG was 34%; and sciatic nerve = 0% (Fig. 6A right, & Fig. 6C). Thus, the PlpCre construct is not active within the GFAP+ nonmyelinating cells in the peripheral nerve. In neurofibromas, months after tamoxifen exposure PlpCre;Nf1fl/fl cells did not express GFAP (Fig. 6C).

There were no EGFP+;p75+ wild type satellite cells within the DRG 4 days after tamoxifen (Fig. 6D). EGFP+;p75+ cells were present in the sciatic nerve (Fig. 6D). EGFP+ cells (59% in DRG, & 41% in sciatic nerve) double-labeled with the Schwann cell and satellite cell marker glutathione synthase (GS) (Fig. 6A right). Thus the PlpCre driver targets myelinating S100β+ Schwann cells within the sciatic nerve, and unidentified p75+ cells. Within the DRG, all EGFP+ cells had the morphology of satellite cells, with distinctive cell processes wrapping around DRG neurons.

Rapid onset of proliferation in Nf1 mutant cells

We noted that the numbers of EGFP+ cells in PlpCre;Nf1fl/fl dorsal root ganglia seemed to increase over time (Fig. 7A). Quantification of EGFP+ cells at 1, 7, and 28 days following tamoxifen injection in the sciatic nerve showed that beginning 1 day after the final tamoxifen injection the numbers of EGFP+ cells were significantly increased in PlpCre;Nf1fl/fl as compared with wild type (Fig. 7B). We monitored cells in the S-phase of the cell cycle with BrdU immunostaining. Many nerve and neurofibroma EGFP+ cells were BrdU+, likely accounting at least in part for the observed increase in cell number (Fig. 7C).

Discussion

In this study we found that both postnatal (P1–3) and adult loss of Nf1 using the PlpCre driver cause GEM-grade I neurofibroma tumor formation. The models differ in the timing of neurofibroma formation, in the size of the neurofibromas generated, and in prevalence of hematopoietic manifestations. We found that myelinating Schwann cells, p75+ cells, and satellite cells are targeted by the inducible PlpCre driver. Our results support previous studies indicating that loss of Nf1 in subpopulations of nerve Schwann cell lineage cells cause neurofibroma formation, and extend these studies by showing that acute Nf1 loss, after organogenesis and cell differentiation, can be tumorigenic.

We identified EGFP+ cells to identify possible tumor cells of origin in the PlpCre model. Tamoxifen exposure induced peripheral nervous system recombination, as judged by EGFP+ cells, in satellite cells in the DRG (S100β+ or GFAP+) with the characteristic morphology of satellite cells, closely wrapping DRG cell bodies. In DhhCre;Nflfl/fl mice, satellite cells do not show recombination, yet neurofibromas form (24). While not definitive, the combination of the two models does not support a role for satellite cells in mouse neurofibroma formation; however, the possibility that satellite cells are important for neurofibroma formation in some settings whereas not in others cannot be excluded.

Most EGFP+ cells in adult peripheral nerves were S100β+ (myelinating) Schwann cells. This result is expected, as endogenous Plp is a characteristic of myelinating Schwann cells in adult peripheral nerve (33). At the EM level, Nf1 loss did not dramatically alter myelination.

Remak bundle disruption is a characteristic feature of all neurofibroma models (22–24, 34). When the nonmyelinated Schwann cell population was examined, no EGFP+;GFAP+ nonmyelinating Schwann cells were identified. These data are surprising as electron microscopy shows disruption of the association between axons and nonmyelinating Schwann cells in the PlpCre;Nf1fl/fl model. We conclude that Nf1 loss within GFAP+ cells is not necessary for Remak bundle disruption in the PlpCre;Nf1fl/fl model.

We identified EGFP+;p75+ cells in peripheral nerve. These may be a subpopulation of GFAP-negative nonmyelinating cells, and/or an as-yet unidentified population(s) in adult peripheral nerve. Zheng and colleagues (2008) proposed that the p75+ nonmyelinating cell population was the cell of origin for neurofibroma formation (23). The present study eliminates the GFAP+ nonmyelinating Schwann cell as the tumor-initiating cell. It is possible that neurofibroma formation results from the p75+/GFAP-negative cells in peripheral nerve, and/or that loss of Nf1 within mature myelinating Schwann cells have noncell autonomous effect(s) that promote tumor formation–similar to the noncell autonomous effect on hematopoietic cells when Nf1 is lost within stromal cells.

Perinatal tamoxifen injection into PlpCre;Nf fl/fl mice resulted in GEM grade I neurofibroma formation that resulted in morbidity at 15 to 23 months old. In contrast, adult loss of Nf1 resulted in large neurofibromas, which caused morbidity beginning 5 months post-tamoxifen introduction. We considered the possibility that recombination occurs in more cells when tamoxifen-induced recombination occurs in adults. However, twice as many cells in sciatic nerve and three times as many in the DRG were EGFP+ in pups as compared with adult mice. Therefore together with previous studies showing that loss of Nf1 in most developing Schwann cells leads to robust neurofibroma formation (22–24) we conclude that cells exist in the adult peripheral nervous system that remain susceptible to neurofibroma formation, and that the susceptible population(s) may be enriched in adult and embryonic nervous systems.

A study submitted by Le and colleagues confirms our observation that neurofibromas can form after perinatal or adult loss of Nf1. However, while they identified small neurofibromas at the thoracic and lumbo-sacral levels after adult loss of Nf1, we generated large neurofibromas throughout the neuroaxis. Some differences between the two studies may account for the slightly different findings. Le and colleagues provided 4 mg tamoxifen (2 mg twice daily) by oral gavage to adult mice for 5 days, whereas we dosed 1 mg twice day by i.p. injection for 3 days. It is also possible that the different phenotypes result from the different tamoxifen-inducible PlpCre driver lines used in the two studies. This difference may well account for the absence of hematopoietic lesions in their system. Most importantly, our data do not support the idea that there is a critical window for neurofibroma formation.

Mast cells, and other hematopoietic cells, showed <1% recombination in PlpCre;Nf1fl/fl mice, yet neurofibroma formation was robust. While Krox20Cre;Nf1fl/fl mice show hyperplasia within the DRG, neurofibroma development required an Nf1+/− background attributed to Nf1+/− mast cells (22, 35). DhhCre;Nf1fl/fl animals develop neurofibromas when only Schwann cell lineage cells are Nf1 mutant (24), and the PlpCre;Nf1fl/fl model is similar in that a heterozygous background is not necessary for neurofibroma formation.

The peripheral blood of PlpCre;Nf1fl/fl mice showed a decrease in monocytes with a relative increase in lymphocytes and polymorphonuclear (PMN). In contrast, Nf1 loss driven by Mx1–Cre causes a mouse disorder similar to human juvenile myelomonocytic leukemia (JMML), with hyperproliferation of all hematopoietic cell types and progressive myeloproliferative disorder (36, 37, 38). Flow cytometry and immunohistochemistry showed that the PlpCre transgene did not cause Nf1 loss in cells of hematopoietic origin. Rather, EGFP+ cells within these tumors had a stromal/mesenchymal appearance, and some double-labeled with Sca-1. EGFP expression did not colocalize with the expression of hematopoietic lineage markers or c-kit (data not shown), indicating that recombined EGFP+ cells had a nonhematopoietic and mesenchymal origin (30, 39).

The idea that Nf1 loss can affect tumorigenesis in a noncell autonomous fashion (e.g., myelinating cells acting upon other cells in the nerve) is thus supported by analogy to the formation of hematopoietic lesions within the PlpCre;Nf1fl/fl animals, in which mutant stromal cells after either perinatal or adult tamoxifen-induced Nf1 loss, cause lymphoid and myeloid proliferation. However we cannot exclude the possibility that neurofibroma formation requires Nf1 loss of function in a small population of stem/progenitor-like cells that remain unidentified by our analysis, or induces expression of chemoattractants that result in massive tissue infiltration by hematopoietic cells. In either event, neurofibroma formation is not restricted to loss of Nf1 in embryonic life, but can be triggered by Nf1 loss throughout life.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

We thank Brian Popko (University of Chicago) for providing the inducible Plp-Cre-ERT mice.

Footnotes

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).